* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download UvA-DARE (Digital Academic Repository) C
Survey
Document related concepts
Lymphopoiesis wikipedia , lookup
DNA vaccination wikipedia , lookup
Social immunity wikipedia , lookup
Hygiene hypothesis wikipedia , lookup
Immune system wikipedia , lookup
Molecular mimicry wikipedia , lookup
Cancer immunotherapy wikipedia , lookup
Polyclonal B cell response wikipedia , lookup
Adoptive cell transfer wikipedia , lookup
Adaptive immune system wikipedia , lookup
Immunosuppressive drug wikipedia , lookup
Transcript
UvA-DARE (Digital Academic Repository) C-type lectin signaling in dendritic cells: molecular control of antifungal inflammation Wevers, B.A. Link to publication Citation for published version (APA): Wevers, B. A. (2014). C-type lectin signaling in dendritic cells: molecular control of antifungal inflammation General rights It is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons). Disclaimer/Complaints regulations If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: http://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible. UvA-DARE is a service provided by the library of the University of Amsterdam (http://dare.uva.nl) Download date: 16 Jun 2017 Pa g hin s a cr rty one. 16 Chapter one one. C-type lectin receptors orchestrate antifungal immunity - an introduction Future Microbiology, 8; 839-854 (2013) – published in modified form Brigitte A. Wevers, Teunis B.H. Department of Experimental Immunology, Academic Medical Geijtenbeek and Sonja I. Center, University of Amsterdam, Amsterdam, NL. Gringhuis Fungal infections are an emerging threat for human health. A coordinated host immune response is fundamental to successful elimination of an invading fungal microbe. A panel of C-type lectin receptors expressed on dendritic cells enables innate recognition of fungal cell wall carbohydrates and tailors adaptive responses by presenting antigen as well as instruction of CD4+ T helper cell fates. Well-balanced T helper cell type 1 and interleukin-17-producing T helper cell responses are crucial in antifungal immunity and facilitate phagocyte clearance of fungal encounters. Strikingly, different classes of fungi trigger distinct sets of C-type lectin receptors to evoke a pathogen-specific T helper response. In this chapter we have outlined the key roles of several C-type lectin receptors during the generation of protective antifungal immunity, with special emphasis on the distinct signaling pathways and transcriptional programs triggered by these receptors, which collaborate to orchestrate polarization of the T helper response. C-type lectin receptors orchestrate antifungal immunity 17 FUNGAL PATHOGENS: FAR BEYOND COMMENSALISM & OPPORTUNISTIC INFECTION Fungi are ubiquitous in the environment. Some fungi, including Malassezia species (spp.), Candida spp., and Pneumocystis jirovecii (formerly Pneumocystis carinii) have successfully established life-long commensal relationships with the human host, and colonize cutaneous and mucosal surfaces without necessarily causing disease1. Even, fungal microbes are being recognized as intestinal commensals (referred to as the mycobiome) that strongly interact with the gut immune system2. Pathogenic fungi take advantage of an altered state of host immunity to cause (lethal) opportunistic infections, with a rapidly growing population of immunosuppressed patients at risk3. Commensal fungal-derived ligands (i.e. β-1,6-glucans) can also drive chronic clonal expansion of mature B cells, and, in doing so, might contribute to the pathogenesis of B cell chronic lymphocytic leukemia (B-CLL)4. Although largely unrecognized, this view of fungal epidemiology is dramatically challenged by the growing incidence of fungal diseases in seemingly healthy individuals. Emerging pathogenic fungi, such as Coccidioides immitis, Histoplasma capsulatum, and Blastomyces dermatitidis, have developed many elaborate mechanisms to overcome host immune surveillance and establish primary and life-threatening infections1. Highly virulent Cryptococcus gattii genotypes have gained recent prominence following a major and ongoing outbreak of fatal cryptococcal meningitis in western North America5. Resisting fungal infection. The human immune system is equipped with effective defense mechanisms to mediate protection to fungal infection, yet activation of these responses requires the coordinated activation and complex interplay of specialized types of immune cells. Hence, host immunity has to accomplish a challenging task: maintaining tissue homeostasis by eradicating invading fungi that can cause harm, while preventing immunopathology and tolerating the commensal fungal strains being important for our health6,7. The human immune system comprises two arms that complement one another: innate immunity (‘natural’ immunity) and adaptive immunity (‘acquired’ immunity). The innate infection8. Skin and epithelial surfaces act as physical barriers of protection, and at mucosal tissues, mucus layers and immunoglobulin A (IgA) secreted by plasma B cells work in concert to prevent tissue invasion by fungal pathogens9-11. Innate effector cells residing in skin and mucosa, such as interleukin (IL)-17-producing innate lymphoid cells (ILCs) and epithelial cells, further contribute by producing antimicrobial peptides12. When fungal pathogens successfully breach host barriers, rapidly recruited phagocytic cells, including neutrophils and macrophages, facilitate immune protection during the earliest stages of infection and mediate local fungal elimination13,14. Despite these effective immune mechanisms, the innate system lacks specificity or the ability to generate immunological memory and life-long protection. These tasks are accomplished by the effector B and T lymphocyte populations from one. system facilitates immediate but non-specific host defense mechanisms against microbial 18 Chapter one Box 1. Pathogen-specific lympocyte populations. 1. Pattern recognition 2. Maturation DAMPs PAMPs Memory cells 3. Migration to central lymphoid organs CD8 IL-2 IL-12 Naive CD8+ T cell Dendritic cell Mature DC Effector CTL Granzyme Perforin IL-2 (TH1) MHC I Cytokines IFN-I CD8 Costimulation Memory cells B cell IL-2 IFN-γ MHC II B cell TH cell (T-bet) IL-12 Naive B cell IL-21 IL-27 TFH cell (Bcl-6) Naive CD4+ T cell IL-23 IL-6 TH17 cell (RoRγT) IL-4 Plasma cell IgG2a IFN-γ (TH1) IL-4 IL-13 Phagocyte activation IL-22 IL-17 IL-1β B cell help Immunity to fungi, intracellular pathogens Immunity to fungi and bacteria Mucosal homeostasis TGF-β TH2 cell (GATA3) Immunity to extracellular parasites T reg (Foxp3) IL-2 TGF-β TGF-β IL-10 Regulation, tolerance IgE Introduction IgG1 IL-4 (TH2) Upon delivery of their cognate antigen by DCs, of the effector or ‘helper’ CD4+ T cell subset in addition to receiving co-stimulatory and family, discovered more than 25 years ago150. cytokine signals, naive T cells in secondary Ever since, numerous other heterogeneous lymphoid organs become activated and Th subsets have been characterized – the differentiate into effector lymphocyte three most prevalent being Th17 cells that populations -each with specific functions and produce IL-17151, T regulatory (Treg) cells33 gene expression programs for appropriate and T follicular helper (Tfh) cells152. Through elimination of different types of microbes. actions of lineage-specific transcription Th1 and Th2 cells are the founding members factors, CD4+ T cells differentiate into effector C-type lectin receptors orchestrate antifungal immunity 19 the adaptive immune system, characterized subsets that secrete restricted patterns of by lineage-specific effector molecules and cytokines and express different chemokine regulate host immunity in a pathogen-specif- type of threat encountered153,154. Th1 cells are dedicated to efficiently combat intracellular bacteria and viruses, by producing IFN-γ, ic manner (Box 1). Not surprisingly therefore, B and T cells play a central role in providing optimal protection to insults by fungal patho- while Th2 cells produce IL-4 and IL-13 for gens15,16, with the fundamental importance defense to extracellular parasites155. The Th17 of effector CD4+ T cells dramatically exem- cell subset, on the other hand, selectively plified by the exceptionally high frequency produces IL-17, providing protection to fungal of life-threatening cryptococcal infections and bacterial threats21. The population of in HIV-1/AIDS patients with declined CD4+ T Treg cells, producing cytokines TGF-β and cell numbers17. IL-10, plays a crucial role in the maintenance of immune homeostasis156. Tfh cells represent another effector CD4+ T cell population with a specialized function: Tfh cells help B cells generate antibody responses to T Two functionally distinct CD4+ T cell subsets are considered key to effective fungal microbe elimination: T helper cell type 1 (Th1) and IL-17-producing T helper (Th17) cell-dependent antigens for clearance of cells. Th1 and Th17 responses can be induced pathogens by phagocytes or the complement in parallel, yet, although still considerably system . Effector CD8 T cells, cytotoxic T uncertain, their degree of contribution is pre- cells -in contrast to helper CD4+ T cells not sumably context dependent, e.g. pathogen- further subdivided- produce multiple effector and/or tissue-specific18: Th1 cells take part molecules, such as perforin and granzyme, in the cellular defense, important during dis- and are specialized in destroying virally seminated disease, and orchestrate optimal 157 + infected cells and/or tumor cells158. About 10% of the effector CD4+ and CD8+ T cells acquire a memory phenotype: quiescent long-lived central memory T cell (Tcm) and macrophage activation, whereas Th17 cells predominantly maintain barrier immunity at mucosal surfaces and act on neutrophils. effector memory T cell (Tem) populations These effector T cells secrete cytokines to that are able to quickly respond to antigen mediate their influence on other immune re-exposure . Memory B cell generation, cells during an antifungal immune response. in addition to isotype switching to IgG, IgA Th1 cells secrete interferon-γ (IFN-γ), which and IgE subtypes, depends on cytokine triggers a plethora of systemic effector mech- present in the local environment (i.e. Th2- anisms, such as antibody class switching to derived IL-4 induces IgE class-switching)160. opsonizing subtypes, upregulation of MHC 159 Although Th cells have been considered terminally differentiated immune cells, this view is considerably challenged in recent molecules for enhanced antigen presentation, and stimulation of macrophage effector years, with many examples of Th cells functions (e.g. production reactive oxygen flexible in their cytokine production profile, intermediates)19. IFN-γ might also directly and hence their effector phenotype153. affect fungal growth, as it inhibits the yeastto-hyphal transition in C. albicans20. The Th17 one. receptors, ensuring tailored responses to the 20 Chapter one effector molecule is an important mediator of tissue inflammation: IL-17 acts on a broad range of immune and non-immune cells and is key to the recruitment, migration and activation of neutrophils21. Th17 cells are also an important source for IL-22, which promotes, together with IL-17, production of protective antimicrobial peptides to mediate mucosal microbial resistance22,23 (Box 1). The protective role of Th17 responses during fungal infection is underscored by the severe recurrent and chronic Candida infections in patients with genetic defects in the Th17 axis, including individuals suffering from chronic granulomatous disease (CGD) and hyper IgE syndrome24-30. Similarly, Th17 cells and IL-17 have been shown to mediate protection in numerous experimental mouse models of fungal infection31,32. Paradoxically, exaggerated antimicrobial Th17 responses are often associated with tissue damage. The magnitude of pathogenic Th17 cell activity as well as unwanted Th17 responses directed against commensal fungi can be kept in check by regulatory T (Treg) cells, the natural gatekeepers of immune homeostasis33. In any case, finely tuned Th1 and Th17 responses probably maximize fungal elimination and, at the same time, minimize host tissue damage during inflammation (recent reviews on this topic have been published elsewhere:16,34,35). Instruction of the adaptive (antifungal) response is coordinated through the actions of specialized antigen-presenting cells (APCs), principally dendritic cells (DCs), which provide all signals necessary for naive T cells to acquire an effector phenotype: T cell receptor (TCR) stimulation, co-stimulation and cytokines36, thereby contributing to T cell-dependent B cell help and generation of antibody responses. DENDRITIC CELLS AND THE GENERATION OF ANTIFUNGAL IMMUNITY DCs reside in the periphery or circulate through blood and monitor for signs of microbial attack, but also host derived danger signals released in response to stress, tissue damage and necrotic cell death. Being specialized in sensing conserved microbial structures termed pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs), through pattern recognition receptors (PRRs), DCs can discriminate between different classes of potential danger37,38. PAMPs refer to molecules associated with pathogenic and non-pathogenic microbes, such as cell wall components and nucleic acids of fungal, bacterial and viral origin, while DAMPs are endogenous molecules released upon stress or damage to the host: amongst others high-mobility group protein 1 (HMGB1), heat-shock proteins (HSPs), extracellular ATP and uric acid crystals. Immature DCs capture Introduction and internalize pathogens or self-molecules, simultaneous encounter with PAMPs/DAMPs induces a cascade of phenotypical changes. This so-called maturation process entails upregulation of costimulatory molecules, lymphoid tissue homing receptor CCR7, and major histocompatibility complex (MHC)-antigen complexes, allowing DCs to activate naive T cells in central lymphoid organs. Responding T cells start to proliferate and differentiate, and, as C-type lectin receptors orchestrate antifungal immunity 21 distinct effector populations, rapidly enter sites of local inflammation were they perform their effector function to aid in pathogen-specific clearance (Box 1). Pattern recognition receptors. Instruction of the adaptive response by DCs is subject to tight regulation, and this process is dependent on a large panel of germ-line encoded PRRs39. DCs express a large variety of membrane- and cytoplasmic-localized PRRs, including the archetypical Toll-like receptors (TLRs) and C-type lectin receptors (CLRs) and NOD-like receptors (NLRs) (further discussed in Box 2). To coordinate DC-induced inflammatory responses, PRRs control four crucial processes. First, PRRs allow DCs to discriminate between different classes of PAMPs and DAMPs, and as such ‘license’ them to drive pathogen-specific responses37. Moreover, DC-expressed PRRs facilitate internalization and processing of pathogen-derived antigens for subsequent antigen presentation in the context of MHC molecules. In addition, a selective set of PRRs induces intracellular signaling for activation of two additional processes: transcriptional activation of a core set of innate response genes, leading to expression of co-stimulatory molecules, chemokines and cytokines40,41; and the assembly of cytosolic protein complexes, inflammasomes, for posttranslational processing of IL-1β family members42 (Figure 1). Since the local cytokine milieu created by dendritic cells is instrumental to the fate lineage decision of differentiating CD4+ T helper cells36,43, PRR-induced signaling is crucial for clonal expansion and differentiation of a responding antigen-primed T cell population. Fundamental to the expression of many inflammatory cytokine and chemokine genes is the activation of the nuclear factor-κ B (NF-κB) family of transcription factors, which are designated as central coordinators of the innate immune response. NF-κB homo- and heterodimers are retained inactive within the cytoplasm by inhibitory proteins of the IκB family. Upon a PRR-mediated signal, the IκB inhibitory complex is degraded, and subsequently initiates release and nuclear translocation of NF-κB dimers44. In addition, PRRs also activate other transcription factors, such as transcription factor activator protein-1 (AP-1) factors (IRFs) for induction of type I interferon (IFN-I) responses. Tailoring T helper responses to fungal infection. The local cytokine milieu created by DCs is instrumental to the fate lineage decision of differentiating CD4+ T helper cells36,43; cytokine actions involve direct induction or repression of a lineage-specific transcription factor or essential growth factor(s). Regarding activation and maintenance of human antifungal T h1 and T h17 effector subsets, several cytokines are considered of crucial importance. T h1 cells differentiate from naive T cells in response to DC-derived IL-1245. IL-12 binding to its cognate receptor (IL12R) on activated CD4+ T cells triggers, via STAT4, transcription of the lineage-specific transcription factor T-bet46, which mediates the one. for expression of cytokines and chemokines, as well as numerous interferon regulatory 22 Chapter one i Pattern recognition Flagellin LPS TLR5 TLR4 ii Antigen presentation Mannose CLRs RLRs ds/ssRNA Phagosytosis MHC I Phagosome NLRs MDP RIG-I MDA5 Surface NOD2 NLRP3 DAMPs Endosome PAMPs Viruses Host cell death Necrosis Sap130 MHC II Dectin-1 Dectin-2 TLRs dsRNA Lysosome Endogenous proteins TLR9 TLR7/TLR8 CpG DNA ssRNA Endosome dsDNA IFI16 MHC II loading Fungi Bacteria TLR3 Mincle AIM2 β-glucan PYHINs MHC I MHC II Proteasome MHC I loading Surface ER Golgi Figure 1. Four principal roles of pattern recognition receptors (PRRs). (i) Innate immune cell-associated PRRs recognize distinct types of pathogen associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs), allowing instruction of tailored adaptive immune responses. Numerous PRR families have been characterized; while some are stationed at cell membranes, such as Toll-like receptors (TLRs) and C-type lectin receptors (CLRs), others are located within the cytosol. RIG-I-like receptors (RLRs), NOD-like receptors (NLRs) and PYHIN sensors. Prominent PRR family members and their cognate ligands are depicted in the figure. (ii) PRRs facilitate internalization and/or processing of peptide-derived antigens for presentation in the context of major histocompatibility (MHC) class I and II I secretion of IFN-γ. Human T h17 cell fate determination involves multiple cytokines: IL-6, IL-23, IL-1β, IL-21 and TGF-β21, although debate continues regarding requirement and primary source of the latter. T h17 development is dependent on transcription factors Introduction STAT3 and RORγt. Signaling by DC-derived IL-6 and IL-23 directly activates STAT3 and subsequently RORγt. IL-21, another STAT3 activator, is expressed by T h17 cell and promotes maintenance of T h17 differentiation via an autocrine route. IL-1β functions during early and late stages of T h17 cell commitment, possibly by counteracting the inhibitory effects of IL-12 and IL-10 on T h17 differentiation47. T h17 cells produce the 23 C-type lectin receptors orchestrate antifungal immunity iii Gene transcription iv Inflammasome activation TLR4 Dectin-1 Dectin-1 CD14 TRIF K+ efflux Myd88 ROS Stress signals IRAK Syk TLR3 Syk PAMPs DAMPs C/B/M scaffold Mytochondria Canonical TRIF NLRP3 Non-cannonical ASC ASC Pro-caspase-8 RIG-I MAVS RIP2 NOD1/2 Pro-caspase-1 MDA5 TBK1 IRFs Type I IFNs TRAFs TAK1 NF-kB AP1 Chemokines Cytokines Antimicrobial peptides Costimulatory molecules Caspase-1 IL-1β Processing IL-18 Caspase-8 Cytokine release Pro-IL-1β Pro-IL-18 molecules to naive CD4+ and CD8+ T cells, respectively. (iii) Several PRRs transduce intracellular signaling upon their activation, leading to transcriptional activation of numerous innate response genes. TRAF adaptor proteins account for integration and diversification of PRR signaling for activation of different transcription factors. (iv) Furthermore, PRRs can mediate the assembly and activation of cytosolic protein complexes -caspase-1-containing canonical or caspase-8-containing non-canonical inflammasomes- for posttranslational processing and maturation of cytokines from the IL-1β family. C/B/M, CARD9-Bcl-10-MALT1; dsDNA/RNA, double-stranded DNA/RNA; MDP, muramyl dipeptide; IFNs, signature cytokines IL-17A (referred to as IL-17), IL-17F and IL-2221 (Box 1, with Figure 1 in Chapter 6 providing a more comprehensive overview). Foxp3-expressing Tregs restrain uncontrolled chronic T h1 and T h17 effector responses deleterious to the host and exist as a mature T cell subpopulation in the periphery (natural (n)Tregs), but can also be induced from naive CD4+ T cells by IL-2. Induced regulatory T cells (iTregs) acquire suppressive activity in response to transforming growth factor-β (TGF-β)48. Thus, DCs are masters in command of an army of lymphocytes and hence shape the adaptive arm of an ensuing antifungal inflammatory response. Depending on the one. interferons; ROS, reactive oxygen species; ss-RNA, single-stranded RNA. 24 Chapter one fungal species encountered as well as the host cell type, specific PRRs will be activated, which elicit distinct downstream signaling events that collectively determine the overall adaptive response tailored to the encountered microbes. SENSING FUNGAL INVASION: C-TYPE LECTIN RECEPTORS TAKE CENTER STAGE The fungal cell wall composition is dynamic and highly variable, yet consists of a multitude of putative and unique PRR ligands49. The core structure is dominated by polysaccharides, comprising mainly β-1,3-glucan, β-1,6-glucan and chitin polymers, surrounded by a layer enriched in mannosylated glycoproteins50. Phospholipomannan, α-glucans, and galactomannan constitute cell wall components found in only a minority of fungi51. Fungal components can be recognized by more than one receptor (e.g. β-glucan recognition by both langerin and dectin-1), resulting in differential responses, and certain PRRs transduce divergent intracellular signaling pathways upon binding distinct ligands; exemplified by the mannose- and fucose-based signaling induced by DC-specific ICAM-3-grabbing non-integrin (DC-SIGN) with pro- or anti-inflammatory outcomes, respectively (further discussed below). Notably, as we demonstrate in Chapter 2 of this thesis, even closely related strains within one taxonomic group can differentially trigger innate receptors on dendritic cells52. In sum, expression of a plethora of innate PRRs permits the host immune system to mount an effective, and above-all, tailored antifungal adaptive response. TLRs, among the most well characterized PRRs, have been assigned function in the anti- fungal immune response. Fatal Aspergillus fumigatus infections observed in Toll-deficient Drosophila provided an initial link between TLR components and antifungal immunity53. In mammalian studies with murine infection models, TLRs were found to have critical roles in both innate recognition and driving protective responses54,55. Strikingly, however, the control of antifungal defense in men is not dominated by any of the TLR members. Humans with genetic defects in the universal TLR adaptor molecule -shared as well by the IL-1 receptor (IL-1R) and IL-18R- MyD88 are highly susceptible to bacterial, but not fungal infections56,57. Strictly under conditions of severe immunosuppression, single nucleotide polymorphisms (SNPs) in human TLR1 and TLR4 genes predispose to infection with fungi57,58. Also, human TLRs are considered incapable to autonomously elicit robust Th17 skewing52. During fungal infection, engagement of most TLRs potentiates strong IL-12p70 production, and thus favors Th1 polarization16,59, although Treg activation by TLR2 is considered an exception60. Murine Introduction TLRs exhibit some potential to augment Th17 responses61, possibly reflected by their ability to activate transcription factor NF-κB subunit c-Rel (discussed below) in some instances, in marked contrast to their counterparts in men52,62. Thus, at least in the human setting, TLRs cannot be held solely responsible for the control of fungal elimination. Presumably, TLRs contribute to fungal binding and operate as co-stimulators that promote or repress signals C-type lectin receptors orchestrate antifungal immunity 25 by other PRRs to shape the overall antifungal response35. Emerging evidence indicates that the control of human antifungal defense is instead dominated by C-type lectin receptors (CLRs; Box 2). Langerin and mannose receptor (MR) are CLR sensors for fungi with endocytic activity and facilitate or, in the case of MR, contribute to phagocytosis of fungal particles63-66. These receptors subsequently direct delivery of the fungal cargo into the appropriate phagosomal route; internalized fungi are either processed for antigen presentation, intracellular NLRP3/caspase-1 inflammasome activation, or, alternatively, for degradation in an attempt to clear the fungal threat. It is becoming evident that several myeloid CLRs act as PRRs that exhibit potential to transduce intracellular signaling to direct transcription of innate response genes40. Among these signaling CLRs, several have been implicated in antifungal immunity, all with distinct mechanisms of action: dectin-1, dectin-2, mincle, and DC-SIGN67-70. Whether langerin and MR, besides promoting fungal uptake and processing, transduce intracellular signaling has not been formally proven. It is, however, likely that both CLRs modulate intracellular signaling indirectly, simply by influencing recruitment of signaling receptors to the phagocytic synapse71. It is of particular interest that CLR signaling through downstream assembly of a complex containing CARD9 is indispensable for the generation of a Th17-dominated response52,72,73, and additionally induces Th1 polarization72,74. Dectin-1 represents the prototype antifungal CLR, which renders DCs fully competent to direct Th1 and Th17 immunity after exposure to fungal β-glucan72,74. Recent genetic studies in humans signify the importance of the dectin-1/CARD9 axis, as signaling defects have been connected to susceptibility to fungal infection. A SNP in dectin-1 (Y238X) that introduces a premature stop codon and prevents functional dectin-1 expression, predisposes to chronic mucocutaneous candidiasis (CMC), due to limited production of IL-17 and low numbers of Th17 cells in peripheral blood75. Even, in patients receiving immunosuppressive medication, the dectin-1 Y238X SNP has been associated with enhanced susceptibility to invasive aspergillosis76-78. Patients with a loss-of-function CARD9 mutation similarly display greatly reduced numbers of circulating Th17 cells and CMC, but with far more severe clinical symptoms and manifestations of lethal models: deficiency of CARD9 or its upstream effector PKC-δ, results in invasive infection and rapid lethality, rather than the lack of dectin-1 alone72,81. In sum, these studies strongly suggest redundancy between CARD9-coupled receptors, whereas CARD9 dysfunction is detrimental for host control of fungal infection. Apart from dectin-1, CLRs dectin-2 and mincle also transmit signals via the CARD9 module73,82. Indeed, dectin-1 is thought to be dispensable for protection of mice against infection with certain subtypes of fungi, with induction of Th17 responses to systemic C. albicans predominated by dectin-2, not dectin-173,83, and likewise, several studies have suggested contribution of mincle to establishment of protective immunity in mice69,84,85. The studies described in this thesis aimed at the functional characterization of both dectin-2 one. systemic disease79,80. This discrepancy has been observed likewise in murine infection 26 Chapter one Box 2. Pattern Recognition Receptors. The innate immune system senses presence of and expression of genes encoding cytokines, potential danger via recognition of molecular chemokines and antimicrobial peptides162. structures unique to different types of microbes Expression of TLRs is cell-type specific. (PAMPs) or damaged-self (DAMPs), through pattern recognition receptors (PRRs)39. Different The C-type lectin receptor (CLR) superfamily PRRs recognize different types of PAMPs, is a large group of proteins characterized while a given pathogen or self-molecule can be by the presence of one or more C-type recognized by multiple PRRs simultaneously. lectin-like domain(s) (CTLDs)163. CLRs This allows PRRs -in specific combinations- to primarily sense carbohydrate moieties instruct the adaptive immune system how to such as mannose and fucose (e.g. by DC- respond best: providing information about SIGN) as well as β-glucan (i.e. by dectin-1) on the initiation, type, duration and magnitude pathogens and host-derived glycoproteins, of the response . Hence, PRRs dictate the but some recognize F-actin filaments (i.e. outcome of an ensuing immune response. DNGR-1)164,165, and ribonucleoproteins82 Numerous classes of PRRs have been identified, released by necrotic host cells (i.e. mincle). functioning in distinct extra- and intracellular CLRs are implicated in cell adhesion and compartments but also cell types. Most communication processes, detection of cell prominent PRR families are listed below, along death, and uptake of (altered-) self and non- with some of their best-studied members: self-antigens145,166. In addition, several CLRs are 161 able to transduce Syk-dependent signaling, Membrane bound receptors. thereby predominating the antifungal immune Toll-like receptors (TLRs), amongst the best- response. This occurs directly through an studied PRRs, are glycosylated type I membrane intracellular ITAM-like domain (i.e. dectin-1), proteins, comprising a leucine-rich repeat or indirectly via association with an ITAM- (LRR) ectodomain for recognition of well- containing adaptor molecule (e.g. dectin-2 defined PAMPs. The 10 known human TLRs and mincle)40, resulting in induction and/or are stationed at the cell surface for sensing modulation of cytokine and type I interferon (myco)bacterial, fungal and parasitic PAMPs (IFN) responses via transcription factors (e.g. lipopolysaccharide from gram-negative NF-κB, IRF1 and IRF5 (findings described bacteria by TLR4 and bacterial lipoproteins in Chapters 2, 3, 4 and 5 of this thesis). Introduction by TLR1/TLR2 and TLR2/TLR6 complexes), or signal from intracellular vesicles upon Cytoplasmic sensors. detection of double-stranded (ds)RNA (TLR3), NOD-like receptors (NLRs) constitutes the single-stranded microbial (ss)RNA (TLR7) or largest family of the cytoplasmic localized CpG-rich methylated microbial DNA (TLR9). PRRs. Similar to the TLR family, NLRs contain TLRs recruit a single or distinct set of Toll-IL-1 LRR motifs for detection of wide variety of receptor TIR-domain-containing adaptor PAMPs and DAMPs, while, in contrast to TLRs, molecules, such as Myd88, TRIF, TIRAP they are expressed by a wide variety of cell and TRAM, to their cytosolic domains for types. A central nucleotide-binding domain induction of downstream signaling events controls NLR oligomerization, while their C-type lectin receptors orchestrate antifungal immunity 27 caspase-recruitment (CARD), pyrin (PYD) and dependent NF-κB activation, but also mediate baculovirus-inhibitory repeat (BIR) domains NLRP3 inflammasome assembly (RIG-I). The facilitate intracellular signal transduction. third RLR family member, LGP2, lacks a CARD The NLR family comprises more than 20 motif for type I IFN signaling and is thought to members, which can be further divided into function as a modulator of RIG-I and MDA5168. NLRPs (previously NALPs) and NODs (also RLR family members are well known for their known as NLRCs). NLRPs are well-known ability to crosstalk with TLRs and other PRRs for their ability to sense viral infection, upon for modulation of adaptive immune responses; which they assemble into cytoplasmic aberrant or dysregulated RLR signaling has protein complexes (inflammasomes) together been linked to development of autoimmunity. with ASC and caspase-1 for maturation of IL-1β cytokines. NLRP3 assembles into The PYHIN protein family is a group of IFN- inflammasome complexes upon indirect inducible proteins, sensing cytosolic dsDNA sensing of a wide variety of microbial and from viruses and bacteria through their PYD endogenous stress signals (i.e. RNA viruses, and/or HIN200 domain(s). AIM2 is a PYHIN bacteria and mitochondria-derived stress member with established PRR function, signals: mROS, mtDNA and cadiolipin), mediating inflammasome assembly but presumably via detecting a commonly induced not gene transcription, while numerous K+ efflux. NLRC4 assembles inflammasomes PYHINs, including IFI16, are putative dsDNA upon recognition of bacterial flagellin and a sensors signaling for STING-dependent type III and IV secretion system components . 167 induction of cytokines and type I IFNs169. Despite containing a CARD-domain, NOD1 and NOD2 are non-inflammasome forming NLRs; NOD1 and NOD2 sense bacterial peptidoglycan motifs and as oligomers associate with adaptor RIP2 for induction of signaling leading to expression of cytokines, chemokines and reactive oxygen species (ROS)42. and MDA5, are a family of DExD/H box RNA helicases, sensing ssRNA and dsRNA from viral origin or processed-self within the cytoplasmic compartment. RIG-I is also capable of sensing dsDNA indirectly, after it has been processed into ssRNA structures via RNA polymerase III (RNAP III). RIG-I and MDA5 signal via an IPS-1 signalosome for induction of type I IFN responses via transcription factors IRF1, IRF3 and IRF7 or cytokines via CARD9/Bcl-10- one. RIG-I-like receptors (RLRs), including RIG-I 28 Chapter one and mincle in human antifungal immunity, and in accordance with the above notions, we provide evidence for key roles for both dectin-2 (Chapter 2) and mincle (Chapters 3, 4 and 5). DC-SIGN signals independent of the CARD9 axis, yet binds several fungi and dynamically regulates assembly of its signalosome to amplify or inhibit T helper polarization86. Thus, multiple CLRs (i.e. dectin-1, DC-SIGN, dectin-2 and mincle) signal collaboratively to yield the most optimal antifungal response; the mechanisms of which are now beginning to be elucidated at the molecular level. The remainder of this chapter focuses on the distinct signaling pathways and transcriptional programs by which CLRs dectin-1, DC-SIGN, dectin-2 and mincle can influence the adaptive outcome of an antifungal response. We explore the hypothesis that, in terms of effector mechanisms, signaling CLRs implicated in human antifungal immunity can be classified into two distinct groups: (i) receptors that act individually and provide all transcriptional signals required to bridge innate and adaptive responses -that is by instructing CD4+ T helper cell polarization, and (ii) receptors that respond cooperatively and modulate signals from other PRRs to fine-tune a particular adaptive response. Dectin-1 is the prototype of an inducer, specialized in directing both Th1 and Th17 cell responses, whereas DC-SIGN is a prominent member of the second class that distinctively modulates Th1 responses. In fact, our current studies (described in Chapters 2, 3, 4 and 5 of this thesis) reveal that CLRs dectin-2 and mincle can also be classified as modulating CLRs. DECTIN-1: THE CORNERSTONE OF HUMAN ANTIFUNGAL IMMUNITY Dectin-1, the central paradigm for a signaling CLR, is expressed primarily in cells from myeloid origin, including DCs, macrophages, monocytes, neutrophils, langerhans cells (LCs) and eosinophils, yet also found on B cells and mouse innate γδ T cells63,87,88. By means of its β-1,3-glucan and β-1,6-glucan carbohydrate specificity, dectin-1 is capable of binding most if not all fungi, due to the abundance of β-glucans (polymers of D-glucose linked by β-glucosidic bonds) in nearly all fungal cell walls50. Ligand binding by dectin-1 occurs in a Ca2+-independent manner, which is divergent from most other CLRs. Studies in both human and mice have documented recognition of numerous pathogenic species by dectin-1, including Aspergillus spp., Candida spp., Coccidiodides spp., capsule-deficient C. neoformans, Fonsecaea pedrosoi, H. capsulatum, and P. jirovecii85,89-95. Also, dectin-1 interacts with myco- Introduction bacteria, albeit via recognition of a yet unknown ligand96,97. Not surprisingly, fungal cell wall β-glucan abundance influences initial innate detec- tion by dectin-1. The opportunistic pathogen C. albicans has a dimorphic appearance, and β-glucans become more accessible during transition from the commensal yeast form into the invasive filamentous form98. Although the ability to undergo phase transition is strong- C-type lectin receptors orchestrate antifungal immunity 29 ly associated with fungal pathogenicity98, the host might use this event to discriminate invasion from colonization and provoke an antifungal inflammatory response99. Dectin-1 presumably is an important player in local tissue immunosurveillance, with commensal fungi being important constituents of the host skin, oral, and gut microbiota2. Interestingly, a dectin-1 gene variant has been associated with aggravation of inflammatory bowel disease (IBD) severity2, suggesting that altered sensing of fungi by dectin-1 contributes to aberrant immune responses in IBD. Nevertheless, some pathogenic fungal strains strategically mask their β-1,3-glucans to prevent immune recognition, even phagocytosis, and succeed in gaining virulence. The immunologically inert capsule of Cryptocuccos spp. and hydrophobic, RodA-rich, layer of Aspergillus conidia are considered most extreme examples100,101. In myeloid cells, dectin-1 transduces downstream signaling via a unique intracellular signaling domain, which delivers activation signals to Src and Syk family kinases. This domain resembles an immunoreceptor tyrosine-based activation motif (ITAM), termed hemITAM, but differs from a conventional ITAM in that it possesses only one of the two Tyrx-x-Leu (YxxL) sequences102. Binding of the tandem Src homology domain 2 (SH2) domains of Syk to dually phosphorylated ITAMs is crucial for Syk activation. Because of its unusual ITAM, dectin-1 is thought to dimerize to provide such a docking site103,104, analogous to the hemITAM-containing receptor Clec-2105. Syk undergoes autophosphorylation at numerous tyrosines upon binding to dectin-1 in order to initiate downstream signal transduction106 (Figure 2). Two related membrane-associated tyrosine phosphatases (i.e. CD45 and CD148) mediate Src family kinase activation, but need to be quickly segregated from the dectin-1 synapse to avoid dephosphorylation of the ITAM tyrosine residues, and permit productive signaling107. Notably, soluble, β-glucan polymers have been found incapable of excluding CD45 and CD148 activity, even though they bind dectin-1107. This may ensure that dectin-1 signaling is activated solely upon encountering an invading fungus, which should be eliminated, and not harmless shed β-1,3-glucan fragments. Dectin-1 mediates phagocytic uptake of fungal particles107, directs fungal destruction (through production of toxic reactive oxygen intermediates) by macrophages, neutrophils of neutrophils (e.g. degranulation)109. In addition to these immediate antimicrobial effector responses, dectin-1 signaling promotes efficient MHC class II presentation of fungal-derived antigens to CD4+ T cells110, and activates nuclear translocation of transcription factor NF-κB to mediate release of innate response mediators that shape the overall adaptive response. Syk-dependent signaling. In human DCs, dectin-1 autonomously orchestrates activation of all NF-κB subunits, through activation of both the classical and noncanonical NF-κB pathways, and accordingly expression of T h1 and T h17 polarizing cytokines52, via induction of two independent signaling pathways. Syk-dependent signaling induces assembly of a trimolecular signaling complex consisting of CARD9, Bcl-10 and MALT1111. one. and DCs108, and controls, via Ca2+-dependent NFAT transcription, the microbicidal activity 30 Chapter one Inducing receptor Modulating receptor Mannose β-glucan Fungi Dectin-1 Fucose DC-SIGN ROS (+ Signal 2) K63 SYK 1 CARD9 Raf-1 NLRP3 ASC Casp 1 Inflammasomes Caspase 8 CARD9 p100 NIK Raf-1 p p p p MALT1 Bcl-10 Caspase 1 CNK1 p Nucleus p p Raf-1 1. Enhanced p65 activity TAK1 LSP1 2 Ras CN K Ra 1 f-1 KS R1 ITAM-like motif y y p p KSR1 LSP1 Virus 2. Reduced transcription IκBα Canonical RelB NF-κB Non canonical MALT1 Bcl-10 Casp 8 C AS p52 RelB inactivation p a p p65 RelB p65 p50 c-Rel IL-1β IL23A NF-κB p50 IL1B IL6 TH17 promoting IL12A IL12B TH1 promoting pro-IL-1β Figure 2. Dectin-1 provides all transcriptional signals to generate Th1- and Th17- polarizing cytokine profiles, while signaling induced by DC-SIGN modulates CD4+ T cell responses. (a) Upon β-glucan sensing, dectin-1 activates two independent signaling cascades that integrate at the level of nuclear factor-kB (NF-κB) activation: Syk- and Raf-1-based pathways. Recruitment of Syk to the phosphorylated (P) dual tyrosine (Y) motifs of dectin-1 facilitates, via an intermediate kinase (possibly PKCδ; not shown), the assembly of a complex consisting of CARD9, Bcl-10 and MALT1. This CARD9/Bcl-10/MALT1 scaffold then presumably undergoes non-degenerative Lys63 (K63)-linked poly-ubiqitination (polyUb)170, which can Introduction be recognized by cofactors such as TAK1 and TRAF proteins. This leads to the activation of IKK subunit β (IKKβ; not shown), which phosphorylates the NF-κB inhibitor protein IkBα, and targets it for proteasomal degradation. Following IkBα degradation, canonical NF-κB subunits (depicted as p65-p50 and c-Rel-p50 dimers) can enter the nucleus, to drive expression of, among other inflammatory mediators, IL-6, IL-1β and IL-23, which induce Th17, and the Th1 polarizing cytokine IL-12p70. Syk-dependent signaling also I C-type lectin receptors orchestrate antifungal immunity 31 initiates an alternative or non-canonical NF-κB pathway that culminates in the activation of RelB: IKKα (not depicted) is activated by NIK, and initiates processing of NF-κB inhibitor p100 into p52, which enters the nucleus together with RelB. CARD9 and Bcl-10 are involved in recruitment of all NF-κB subunits, whereas the paracaspase MALT1, through its proteolytic activity, strictly targets c-Rel. Besides initiation of canonical and non-canonical NF-κB signaling (1), Syk mediates generation of reactive oxygen species (ROS) production for NLRP3/caspase-1 inflammasome activation (2). Raf-1-dependent signaling culminates in phosphorylation at serine 276 (Ser276) and acetylation (a) of p65. Phopshorylated p65 restrains RelB activity, while acetylated p65 has prolonged nuclear activity and enhances transcription of IL6 and IL12 genes. The CARD9/Bcl-10/MALT1 scaffold also initiates formation of an alternative caspase-8 inflammasome in which the CARD9/Bcl-10/MALT1 triad is linked to caspase-8 and the adaptor protein ASC for proteolytic processing of pro-IL-1β. (b) DC-SIGN signaling is ligand-specific and affects Th1 polarization. (left) A pre-assembled trimeric complex consisting of KSR1, CNK and Raf-1 is, via the adaptor molecule LSP1, constitutively associated with the cytoplasmic tail of DC-SIGN. Binding of mannose-containing pathogens (such as fungi) to DC-SIGN induces activation of the serine/threonine kinase Raf-1, which in turn mediates phosphorylation and acetylation of NF-κB subunit p65. These modifications prolong the nuclear activity of p65, resulting in increased transcription rates at specific genes, including those encoding IL-12p70, crucial for antifungal Th1 polarization. (right) Fucose-containing pathogens trigger an alternative, and LSP1-dependent, pathway, accompanied by disassembly of the KSR1/CNK/Raf-1 triad. Activation of this cascade attenuates pro-inflammatory cytokine production, thus negatively affects Th1 responses, via a yet unknown mechanism. Please note that the example given here concerns dectin-1 as an inducer of antifungal Th1 and Th17 responses, but DC-SIGN can influence antifungal responses induced by any other innate receptor. Downstream intermediate PKCδ is likely to couple Syk activity directly to CARD9 phosphorylation and recruitment81. The CARD9/Bcl-10/MALT1 scaffold subsequently activates oligomerization of the IκB kinase (IKK) complex to allow nuclear translocation of canonical NF-κB subunits p65 and c-Rel111. It is not entirely clear how the CARD9/ Bcl-10/MALT1 scaffold targets assembly of the IKK complex, but it presumably occurs indirectly and involves cofactors such as TRAF proteins (e.g. TRAF2 and TRAF6)112 via CARD9 leads to transcription of genes such as IL1B, IL6, IL23A (encoding the p19 subunit of IL-23), IL12A (IL-12p35), and IL12B (IL-12/IL-23 p40 subunit)74, promoting both T h1 and T h17 responses52,74. Unlike most PRRs, dectin-1-Syk signaling simultaneously activates the noncanonical NF-κB subunit RelB, which involves NIK. This pathway partly antagonizes responses induced by dectin-1 through classical NF-κB signaling, since RelB suppresses IL12B and IL1B transcription, and hence p65- and c-Rel-mediated IL-12p70 and pro-IL-1β expression, by preventing RNA polymerase II recruitment74 (Figure 2). Strikingly, dectin-1 activates a second pathway through Raf1 that integrates with Syk-dependent signaling at the level of NF-κB activation, and is crucial for further fine-tuning of cytokine transcription by dectin-1. one. and TAK1, analogous to TCR/BCR signaling81,112. Classical NF-κB signaling by dectin-1 32 Chapter one The road to Th17 induction via MALT1. As mentioned, dectin-1 is a PRR specialized in propagation of T h17 responses. Recent studies have given major new insights into two molecular processes utilized by dectin-1 in human dendritic cells to drive this T h17polarizing cytokine profile. These involve alternative processing of pro-IL-1β 97 and, as our data in Chapter 2 of this thesis point out, selective activation of NF-κB subunit c-Rel52. Strikingly, both rely on the MALT1 scaffold protein, underlining a dual role for MALT1 in shaping T h17 immunity by dectin-197. MALT1 has been found crucial for pro-IL-1β processing via a noncanonical caspase-8 inflammasome. Production of bioactive IL-1β is strictly regulated and requires proteolytic processing of pro-IL-1β, formerly thought solely attributable to the NLRP3/caspase-1 inflammasome. Indeed, internalization of some pathogenic fungi by dectin-1 and subsequent Syk-dependent signaling triggers a conventional NLRP3/caspase-1 inflammasome for the processing of inactive pro-IL-1β into its 17-kDa mature form. Dectin-1, however, directs formation of an alternative noncanonical caspase-8 inflammasome for pro-IL-1β cleavage, without interference of additional cytosolic sensors. This inflammasome consist of the CARD9/Bcl-10/MALT1 scaffold linked to caspase-8 and the adaptor protein ASC, and is assembled in response to all (fungal-) pathogens bound by dectin-197. Indirectly, MALT1 has a crucial and indispensable role in caspase-8 inflammasome activity, given that it directly interacts (presumably dimerization through their respective caspase domains113) with caspase-8 to prevent autocleavage of caspase-8, which would lead to apoptosis. The intermediate processing of caspase-8 allows targeted processing of IL-1β97 (Figure 2). Thus, MALT1 enables dectin-1 to autonomously elicit IL1B transcription as well as rapid IL-1β maturation, to orchestrate induction of Th17 immunity. In addition, as we demonstrate in Chapter 2 of this thesis, MALT1 has a specialized function in regulating expression of Th17-polarizing cytokines IL-23p19 and pro-IL-1β. Specifically, we demonstrate that MALT1, through the selective targeting of NF-κB subunit c-Rel, controls a transcriptional subprogram for efficient induction of IL23A and IL1B gene transcription52, indicating that MALT1 activity is crucial for optimal Th17 effector responses induced by dectin-1; a notion further discussed in Chapter 6. Fine-tuning T helper responses by Raf-1. Dectin-1 is also capable of relaying YxxL- and Syk-independent signaling via the serine/threonine kinase Raf-174, a pathway originally identified downstream of DC-SIGN (further described later on) 62. Dectin-1-mediated Raf-1 activation induces selective phosphorylation and subsequent acetylation of p65, Introduction which has two important functional consequences. First, phosphorylated p65 can restrain RelB into inactive p65-RelB dimers to partially reverse the repressing effects of RelB on IL12B and IL1B transcription. RelB is, however, not completely sequestered and neutralized by p65; residual RelB can induce moderate production of T h2-related chemokines CCL17 and CCL22. Acetylation of p65 typically prolongs its activity and C-type lectin receptors orchestrate antifungal immunity 33 transcription rate, and, downstream dectin-1, results in enhanced expression of IL6, IL12A and IL10 74 (Figure 2). Overall, the Raf-1 pathway permits and fine-tunes induction of T h1 and T h17 immunity by dectin-1-Syk signaling by balancing p65 and RelB activities. Clearly, dectin-1-induced NF-κB activation is subject to tight regulation by distinct pathways which are induced separately but are cooperating at multiple layers to shape the overall immune response. DC-SIGN: LIGAND-SPECIFIC SIGNALING FOR TH1 MODULATION DC-SIGN is a striking example of a signaling CLR that affects adaptive responses induced by other PRRs at the level of NF-κB activation. Activation of DC-SIGN signaling can have profound effects on immune responses directed against several pathogens (e.g. mycobacteria)86, and is even exploited by HIV-1 to facilitate productive DC infection and subsequent in trans infection of T cells - the primary HIV-1 target cells114. Although the role of DC-SIGN in the host defense against fungi has been studied less extensively, DC-SIGN has the potential to modulate antifungal effector mechanisms when activated by fungal pathogens. Most notably, the immunological outcome is dependent on the carbohydrate composition of the ligand involved; DC-SIGN transduces divergent signaling cascades upon ligation with mannose- or fucose- containing ligands. DC-SIGN is predominantly expressed on DC subsets, yet also found on a subpopulation of macrophages115. Tetrameric surface expression enables DC-SIGN binding to mannose, fucose, N-acetyl-glucosamine (GlcNAc) and mannan moieties with high-avidity, which occurs in a Ca2+-dependent manner116. DC-SIGN is involved in the recognition of endogenous carbohydrates (e.g. ICAM-3 on T cells) as well as ligands derived from numerous pathogens, including viruses, bateria, helminths, and fungi117. DC-SIGN harbours several internalization motifs in its cytoplasmic tail118, which allow for robust endocytic activity and fungal uptake; among the fungal pathogens bound via exposed mannose residues are C. albicans, Despite bearing a classical intracellular YxxL motif, DC-SIGN is an exceptional signaling CLR in that it does not transduce downstream signals via the Syk-CARD9 axis, and, moreover, is incapable of activating transcriptional programs (e.g. cytokine expression) on its own62. A pre-assembled complex consisting of KSR1, CNK and Raf-1 is constitutively attached to the cytoplasmic domain of DC-SIGN via the adaptor molecule LSP186. Mannose-containing pathogens such as mycobacteria and HIV-1, and most likely comprising several species of fungi, induce Raf-1 activation upon DC-SIGN binding. Signaling downstream Raf-1 then leads to modulation of the transcriptional activity of NF-κB subunit p65, yet only when nuclear translocation of p65 is induced by any other PRR. Raf-1 phosphorylates p65 at serine (Ser) 276 and controls subsequent acetylation of p65 by two histone acetyltransferases: CBP and one. A. fumigatus, capsule-deficient C. neoformans, and Chrysosporium tropicum70,119,120. 34 Chapter one p300. These post-translational modifications functionally affect p65, defined by prolonged nuclear activity and increased transcription rates particularly at the IL12A, IL12B, and IL6 genes62. Thus, depending on the PRR coactivated by the fungus, triggering of DC-SIGN/ Raf-1 signaling by mannose-expressing fungi will increase expression of IL-12p70, crucial for activation of a Th1 polarization program (Figure 2). DC-SIGN-Raf-1 signaling can also abolish the RelB-dependent suppression of IL12B and IL1B transcription and thereby directly influence Th17 immunity, as would be the case when dectin-1 is co-ligated. In contrast, in response to fucose-bearing ligands (i.e. the gastric pathogen Helicobacter pylori and endogenous Lewis antigens) the KSR1/CNK/Raf-1 triad is selectively disassembled from the cytoplasmic tail of DC-SIGN for initiation of an alternative, and Raf-1-independent, signaling cascade that attenuates production of pro-inflammatory cytokines (Figure 2), with the exact molecular mechansims still being elusive86. This modulatory pathway can potentially be targeted by pathogenic fungal strains for evading an immune response, with fucosylated moieties found in selected species of fungi121. Overall, these findings support the hypothesis that carbohydrate-specific signaling by DC-SIGN can effectively modulate an antifungal immune response induced by other PRRs, and customize it to the fungal strain encountered. Whether DC-SIGN indeed affects fungus-specific T helper cell differentiation awaits further investigation. DECTIN-2 Apart from being typified as a LC marker122,123, human dectin-2 is more generally expressed among cells from myeloid origin, specifically DC subtypes, macrophages, monocytes, and neutrophils14,52,123. Dectin-2 possesses a typical Ca2+-dependent CTLD with affinity for high-mannose structures (Man9GlcNAc2)124. Consistently, dectin-2 has been implicated in recognition of α-mannose from a number of fungi: Candida spp., A. fumigatus, capsule-deficient C. neoformans, H. capsulatum, Microsporum audouinii, Malassezia spp. Paracoccoides brasiliensis, and Trichophyton rubrum52,68,124-126, but also unknown components from house dust mite allergens, the parasitic worm Schistosoma mansoni, and Mycobacterium tuberculosis124,125,127. In contrast to what its name suggest, dectin-2 is only 27% homologous to dectin-1. The short cytoplasmic tail of dectin-2 lacks an obvious signaling motif but associates with the ITAM-bearing Fc receptor common-γ (FcRγ) chain for signal transduction and solid cell surface expression68. In addition, dectin-2 likely forms a heterodimeric PRR with another, Introduction less well-characterized, ITAM-coupled CLR128,129: MCL (also referred to as dectin-3)130. Their respective homodimers were found to bind α-mannose structures less efficiently, suggesting that MCL acts as a subunit of a high-affinity PRR complex for sensing fungal infection130. Engagement of dectin-2 induces phosphorylation of the dual tyrosine residues within the coupled FcRγ ITAM and, subsequently, recruitment of Src family kinases and Syk68,73. Syk C-type lectin receptors orchestrate antifungal immunity 35 activation by dectin-2 is a prerequisite not only for CARD9-dependent induction of NF-κBmediated gene transcription, but also for activation of MAP kinase pathways83. Similar to dectin-1 signaling, canonical NF-κB activation by dectin-2 requires assembly of the CARD9/ Bcl-10/MALT1 scaffold73, and probably involves many of the same intermediate players, including TRAFs and TAK181. However, as our data in Chapter 2 of this thesis demonstrate, human DC-expressed dectin-2, in stark contrast to dectin-1, does not equally activate all NF-κB subunits. We demonstrate that dectin-2-Syk signaling culminates in selective c-Rel activation via MALT1 for selective production of Th17-polarizing cytokines IL-1β and IL-23 subunit p19 (IL-23p19). Thus, dectin-2 is a representative of a class of signaling CLRs that modulate antifungal immunity; the implications for Candida albicans-specific Th17 responses are further discussed in Chapter 6. Interestingly, dectin-2 has been assigned function in the (dys-) regulation of pulmonary Th2 responses in a mouse allergy model, further emphasizing a role for dectin-2 as a modulator of T helper cell polarization. Through Syk-dependent generation of proinflammatory lipid mediators, such as cysteinyl leukotrienes, dectin-2 signaling contributes to pathological airway inflammation in response to extracts derived from house dust mite and A. fumigatus125,131. Th2 immunity, characterized by alternatively activated macrophages and antibody class switching to non-opsonizing and IgE subclasses, controls parasitic infections but is considered deleterious during the course of fungal infection16. A. fumigatus is an exceptional fungus in being a successful opportunistic pathogen and major allergen101, and might therefore utilize sophisticated strategies to avoid destruction. It remains to be determined whether dectin-2 signaling in general contributes to Th2-biased immunity, given its ability to recognize the parasite S. mansoni, and whether dectin-2, in doing so, is targeted by (virulent) fungal strains for immune evasion. However, consistent with the immune modulatory functions of dectin-2, this might in fact be dictated by the additional PRR(s) co-activated by these fungi. Mincle expression predominates on myeloid cell types, including DCs, macrophages and neutrophils, yet is also found on B cells132. Through recognition of an ill-defined mannose-rich ligand, possibly α-mannose, present on glycolipids, mincle recognizes several pathogens from fungal origin, including Candida spp., Malassezia spp., F. pedrosoi69,84,126,133. Additionally, mincle exhibits potential to bind the mycobacterial glycolipid trehalose-6,6-dimycolate (TDM, also known as mycobacterial cordfactor), as well as its synthetic derivate TDB (trehalose-6,6-dibehenate), both well-known for their therapeutic adjuvancy134,135. As such, mincle is held responsible for the Th1/Th17 adjuvancy of TDM and TDB in mice135,136, yet it is still unclear whether mincle has a role in controlling Mycobacterium tuberculosis infection137,138. one. MINCLE 36 Chapter one In addition, mincle has been implicated in anti-bacterial defenses; mincle appears to have a non-redundant role in the protective response to Klebseilla pneumonia infection in mice, by preventing hyperinflammation, although direct binding of mincle to these bacteria has not been proven139. While mincle is not directly involved in the phagocytic engulfment of particulate cargo82,84, it does localize to the phagocytic synapse when macrophages interact with C. albicans84. Mincle possesses a single extracellular CRD for the recognition of microbial carbo- hydrates in a Ca2+-dependent fashion69,140. The mincle CRD contains a primary glutamic acid/proline-asparagine (EPN) motif, predictive for its mannose specificity, flanked by a hydrophobic groove, for binding branched acyl chains (fatty acids)140,141. This additional binding domain probably fine-tunes the specificity of mincle towards the sugar-proximal parts of lipid-moieties. Its cytoplasmic tail is devoid of any classic signaling motifs, and via a positively charged arginine residue, mincle associates with the FcRγ chain for transduction of ITAM-coupled signaling, in analogy to dectin-282. Moreover, and similar to dectin-2, mincle heterodimerizes with MCL for cell surface expression142. Although structural analyses have demonstrated that MCL also contains a hydrophobic domain for binding the branched acyl chains present in TDM and TDB, its CRD lacks a primary EPN motif140, critical for TDM binding by mincle134. Given that MCL binds TDM with much lower affinity142, and that heterodimer formation with MCL is not unique to mincle, the mincle/MCL interaction possibly confers additional function to each of the molecules. Most likely, heterodimerization with MCL enhances the ligand binding affinity, allowing mincle to detect small numbers of glycolipids on the fungal surface. Furthermore, coupling to MCL might ensure efficient signal transduction, and could also reflect a mechanism by which mincle acquires endocytic receptor function143, with MCL demonstrated to exhibit endocytic activity128, in stark contrast to mincle82,84. Whether mincle and MCL indeed function cooperatively, also in the context of human antifungal immunity, remains to be demonstrated. Next to its role as a microbial sensor, mincle is involved in the innate recognition of dam- aged-self. As such, mincle binds the ribonuceoprotein SAP-130, derived from necrotic cells that have lost their membrane integrity82. The mincle-mediated response to damaged-self comprises infiltration of neutrophils for cellular clean-up82, with a possible role for mincle in the induction of pathological inflammatory events following ischemic stroke144. Strikingly, mincle recognizes SAP-130 in the absence of Ca2+ and independent of the CRD residues involved in fungal binding82, implying dual ligand specificity. Also, the proinflammatory response to necrotic cell death by mincle can be considered a sterile reaction, linked to tis- Introduction sue repair, rather than infection control and full-blown activation of adaptive immunity145. It remains to be established if mincle, analogous DC-SIGN, transduces divergent signaling pathways to influence these distinct proinflammatory processes, or whether this is actually dependent on the presence or absence of co-stimulated PRRs. Ligation of murine mincle activates the highly conserved Syk-CARD9 signaling axis81,82,134, C-type lectin receptors orchestrate antifungal immunity 37 whereas its activity in the context of murine Fonsecaea infection has been shown inadequate for induction of protective antifungal responses85. Although costimulation of other PRRs was demonstrated sufficient for subsequent clearance of the pathogen, these protective responses were dependent on mincle signaling as well, indicative of a modulatory function for mincle during establishment of antifungal immunity85. Our work on the functional characterization of human mincle described in this thesis confirmed and extended these previous findings, by demonstrating that mincle-dependent Syk signaling in human DCs culminates in assembly of the complete CARD9/Bcl-10/MALT1 signaling module (Chapter 3). Most strikingly, though, we found that mincle does not signal for NF-κB activation (Chapter 4), and that mincle does not exhibit the potential to activate cytokine responses on its own (Chapters 3 and 4). Instead, our data uncover an alternate mechanism of action: mincle couples Syk-CARD9 signaling to a PI(3)K-PKB pathway for modulation of cytokine responses induced by other PRRs (Chapters 3, 4 and 5), having serious implications for the ability of human DCs to promote concurrent Th1 and Th17 responses during fungal infection, an issue further discussed in Chapter 6 of this thesis. CONCLUDING REMARKS AND OUTLOOK As defined and discussed here, immunity to fungal infection is orchestrated by multiple CLRs expressed on dendritic cells. Two types of antifungal CLRs can be recognized: (i) receptors that evoke a T helper polarization program autonomously and (ii) receptors that modulate signals from other receptors to fine-tune a particular response. Unbalanced inflammation can have deleterious effects on host immunity, and it is becoming evident that these types of CLRs together transduce collaborative signaling to ensure tightly controlled and tailored effector responses upon fungal intrusion. Further characterization of the molecular mechanisms by which CLRs control these processes may therefore give important insights into defense mechanisms deployed against pathogenic fungi, and will facilitate tailor made Antiviral effectors for antifungal control? Our understanding of antifungal immune responses has been expanded recently by studies supporting a possible role for type I IFNs -classic antiviral effectors- during fungal-induced inflammation. Type I IFNs are produced by murine DCs in response to cytoplasmic fungal nucleic acids 146,147, but also it has been demonstrated that murine dectin-1 directly signals for IFN-β expression148, though precise functional contribution to the overall antifungal immune response remains enigmatic. Moreover, defective expression of human type I IFN genes is associated with susceptibility to CMC, albeit through unknown mechanisms149. Our study described in Chapter 5 corroborates these initial findings by providing evidence one. vaccines and novel targets to combat fungal infections. 38 for contribution of a type I IFN response to Chapter one REFERENCES human antifungal inflammation, which also sheds new light on the molecular prin- 1. Brown, G.D., Denning, D.W., ciples of CLR cooperation. Gow, N.A., Levitz, S.M. et al. Hidden killers: human fungal infections. Sci. Transl. Med. 4, 165rv113 (2012). 2. Iliev, I.D., Funari, V.A., Taylor, K.D., Nguyen, Q. et al. Interactions between commensal fungi and the C-type lectin receptor Dectin-1 influence colitis. Science 336, 1314-1317 (2012). 3. Miceli, M.H., Diaz, J.A. & Lee, S.A. Emerging opportunistic yeast infections. Lancet Infect. Dis. 11, 142-151 (2011). 4. Hoogeboom, R., van Kessel, K.P., Hochstenbach, F., Wormhoudt, T.A. et al. A mutated B cell chronic lymphocytic leukemia subset that recognizes and responds to fungi. J. Exp. Med. 210, 59-70 (2013). 5. Kronstad, J.W., Attarian, R., Cadieux, B., Choi, J. et al. Expanding fungal pathogenesis: Cryptococcus breaks out of Introduction the opportunistic box. Nat. Rev. Microbiol. 9, 193-203 (2011). 6. Sommer, F. & Backhed, F. The gut microbiota--masters of host 39 C-type lectin receptors orchestrate antifungal immunity 18. van de Veerdonk, F.L. & development and physiology. Leibundgut-Landmann, S. Nat. Rev. Microbiol. 11, 227-238 Cutting Edge: IL-17-Secreting Netea, M.G. T-cell Subsets and (2013). Innate Lymphoid Cells Are Antifungal Host Defenses. Essential for Host Defense Current fungal infection reports 7. Iliev, I.D. & Underhill, D.M. against Fungal Infection. J. 4, 238-243 (2010). Striking a balance: fungal Immunol. 190, 521-525 (2013). 19. Schroder, K., Hertzog, P.J., commensalism versus pathogenesis. Curr. Opin. 13. Brown, G.D. Innate antifun- Microbiol. 16, 366-373 (2013). gal immunity: the key role of Interferon-gamma: an overview phagocytes. Annu. Rev. of signals, mechanisms and 8. Hooper, L.V. & Macpherson, Immunol. 29, 1-21 (2011). A.J. Immune adaptations that Ravasi, T. & Hume, D.A. functions. J. Leukoc. Biol. 75, 163-189 (2004). maintain homeostasis with the 14. Taylor, P.R., Roy, S., Leal, S.M., intestinal microbiota. Nat. Rev. Jr., Sun, Y. et al. Activation of Immunol. 10, 159-169 (2010). neutrophils by autocrine Prostaglandin E2 enhances and IL-17A-IL-17RC interactions gamma interferon inhibits germ 20. Kalo-Klein, A. & Witkin, S.S. 9. de Repentigny, L., Aumont, F., during fungal infection is tube formation in Candida Bernard, K. & Belhumeur, P. regulated by IL-6, IL-23, albicans. Infect. Immun. 58, Characterization of binding of RORgammat and dectin-2. Nat. 260-262 (1990). Candida albicans to small Immunol. (2013). 21. Korn, T., Bettelli, E., Oukka, M. intestinal mucin and its role in adherence to mucosal 15. Casadevall, A. & Pirofski, L.A. & Kuchroo, V.K. IL-17 and Th17 epithelial cells. Infect. Immun. Immunoglobulins in defense, Cells. Annu. Rev. Immunol. 27, 68, 3172-3179 (2000). pathogenesis, and therapy of 485-517 (2009). fungal diseases. Cell host & microbe 11, 447-456 (2012). Cleavage of E-cadherin: a 22. De Luca, A., Zelante, T., D’A ngelo, C., Zagarella, S. et al. mechanism for disruption of 16. Wuthrich, M., Deepe, G.S., Jr. IL-22 defines a novel immune the intestinal epithelial barrier & Klein, B. Adaptive immunity pathway of antifungal by Candida albicans. Transl. to fungi. Annu. Rev. Immunol. resistance. Mucosal Immunol. 3, Res. 149, 211-222 (2007). 30, 115-148 (2012). 361-373 (2010). 11. van Spriel, A.B., Sofi, M., 17. Park, B.J., Wannemuehler, 23. Kolls, J.K., McCray, P.B., Jr. & Gartlan, K.H., van der Schaaf, A. K.A., Marston, B.J., Govender, N. Chan, Y.R. Cytokine-mediated et al. The tetraspanin protein et al. Estimation of the current regulation of antimicrobial CD37 regulates IgA responses global burden of cryptococcal proteins. Nat. Rev. Immunol. 8, and anti-fungal immunity. PLoS menigitis among persons living 829-835 (2008). Pathog. 5, e1000338 (2009). with HIV/AIDS. AIDS 23, 525-530 (2009). 24. Milner, J.D., Brenchley, J.M., 12. Gladiator, A., Wangler, N., Laurence, A., Freeman, A.F. et al. Trautwein-Weidner, K. & Impaired T(H)17 cell differentia- one. 10. Frank, C.F. & Hostetter, M.K. 40 Chapter one tion in subjects with autosomal 29. Kisand, K., Boe Wolff, A.S., 35. LeibundGut-Landmann, S., dominant hyper-IgE syndrome. Podkrajsek, K.T., Tserel, L. et al. Wuthrich, M. & Hohl, T.M. Chronic mucocutaneous Immunity to fungi. Curr. Opin. candidiasis in APECED or Immunol. 24, 449-458 (2012). Nature 452, 773-776 (2008). 25. Ma, C.S., Chew, G.Y., Simpson, thymoma patients correlates N., Priyadarshi, A. et al. with autoimmunity to 36. Zhu, J., Yamane, H. & Paul, Deficiency of Th17 cells in Th17-associated cytokines. J. W.E. Differentiation of effector hyper IgE syndrome due to Exp. Med. 207, 299-308 (2010). CD4 T cell populations (*). Annu. mutations in STAT3. J. Exp. Med. 205, 1551-1557 (2008). Rev. Immunol. 28, 445-489 30. Puel, A., Cypowyj, S., (2010). Bustamante, J., Wright, J.F. et al. 26. Liu, L., Okada, S., Kong, X.F., Chronic mucocutaneous 37. Iwasaki, A. & Medzhitov, R. Kreins, A.Y. et al. Gain-of- candidiasis in humans with Regulation of adaptive function human STAT1 inborn errors of interleukin-17 immunity by the innate mutations impair IL-17 immunity. Science 332, 65-68 immune system. Science 327, immunity and underlie chronic (2011). 291-295 (2010). 31. Conti, H.R. & Gaffen, S.L. Host 38. Matzinger, P. The danger responses to Candida albicans: model: a renewed sense of self. Th17 cells and mucosal Science 296, 301-305 (2002). mucocutaneous candidiasis. J. Exp. Med. 208, 1635-1648 (2011). 27. van de Veerdonk, F.L., candidiasis. Microbes Infect. 12, Plantinga, T.S., Hoischen, A., 518-527 (2010). Smeekens, S.P. et al. STAT1 Medzhitov, R. Innate immune mutations in autosomal 32. Hernandez-Santos, N. & recognition. Annu. Rev. dominant chronic mucocutane- Gaffen, S.L. Th17 cells in Immunol. 20, 197-216 (2002). ous candidiasis. N. Engl. J. Med. immunity to Candida albicans. 365, 54-61 (2011). Cell host & microbe 11, 425-435 40. Geijtenbeek, T.B. & Gringhuis, (2012). S.I. Signalling through C-type 28. Puel, A., Doffinger, R., lectin receptors: shaping Natividad, A., Chrabieh, M. et al. 33. Littman, D.R. & Rudensky, immune responses. Nat. Rev. Autoantibodies against IL-17A, A.Y. Th17 and regulatory T cells Immunol. 9, 465-479 (2009). IL-17F, and IL-22 in patients in mediating and restraining with chronic mucocutaneous inflammation. Cell 140, 845-858 41. Takeuchi, O. & Akira, S. candidiasis and autoimmune (2010). Pattern recognition receptors polyendocrine syndrome type Introduction 39. Janeway, C.A., Jr. & and inflammation. Cell 140, I. J. Exp. Med. 207, 291-297 34. Romani, L. Immunity to (2010). fungal infections. Nat. Rev. Immunol. 11, 275-288 (2011). 805-820 (2010). 42. Philpott, D.J., Sorbara, M.T., Robertson, S.J., Croitoru, K. & 41 C-type lectin receptors orchestrate antifungal immunity Girardin, S.E. NOD proteins: 48. Curotto de Lafaille, M.A. & sour treat for toll-like receptors. regulators of inflammation in Lafaille, J.J. Natural and Front. Cell. Infect. Microbiol. 2, 142 (2012). health and disease. Nat. Rev. adaptive foxp3+ regulatory T Immunol. 14, 9-23 (2014). cells: more of the same or a division of labor? Immunity 30, 55. Wuthrich, M., Gern, B., Hung, 43. Kapsenberg, M.L. Dendritic- 626-635 (2009). C.Y., Ersland, K. et al. Vaccine- cell control of pathogen-driven induced protection against 3 T-cell polarization. Nat. Rev. 49. Latge, J.P. Tasting the fungal systemic mycoses endemic to Immunol. 3, 984-993 (2003). cell wall. Cell. Microbiol. 12, North America requires Th17 863-872 (2010). cells in mice. J. Clin. Invest. 121, 44. Ghosh, S., May, M.J. & Kopp, 554-568 (2011). E.B. NF-kappa B and Rel 50. Bowman, S.M. & Free, S.J. proteins: evolutionarily The structure and synthesis of 56. von Bernuth, H., Picard, C., conserved mediators of the fungal cell wall. Bioessays Jin, Z., Pankla, R. et al. Pyogenic immune responses. Annu. Rev. 28, 799-808 (2006). Immunol. 16, 225-260 (1998). bacterial infections in humans with MyD88 deficiency. Science 51. Levitz, S.M. Innate recogni45. Moser, M. & Murphy, K.M. tion of fungal cell walls. PLoS Dendritic cell regulation of Pathog. 6, e1000758 (2010). 321, 691-696 (2008). 57. Plantinga, T.S., Johnson, M.D., Scott, W.K., van de Vosse, E. et al. Th1-Th2 development. Nat. Immunol. 1, 199-205 (2000). 52. Gringhuis, S.I., Wevers, B.A., Toll-like receptor 1 polymor- Kaptein, T.M., van Capel, T.M. et phisms increase susceptibility 46. Thieu, V.T., Yu, Q., Chang, H.C., al. Selective C-Rel activation via to candidemia. J. Infect. Dis. 205, Yeh, N. et al. Signal transducer MALT1 controls anti-fungal 934-943 (2012). and activator of transcription 4 T(H)-17 immunity by dectin-1 is required for the transcription and dectin-2. PLoS Pathog. 7, 58. Bochud, P.Y., Chien, J.W., factor T-bet to promote T helper e1001259 (2011). Marr, K.A., Leisenring, W.M. et al. Immunity 29, 679-690 (2008). 53. Lemaitre, B., Nicolas, E., phisms and aspergillosis in Michaut, L., Reichhart, J.M. & stem-cell transplantation. N. 47. Zielinski, C.E., Mele, F., Hoffmann, J.A. The dorsoven- Engl. J. Med. 359, 1766-1777 Aschenbrenner, D., Jarrossay, D. tral regulatory gene cassette (2008). et al. Pathogen-induced human spatzle/Toll/cactus controls the Th17 cells produce IFN-gamma potent antifungal response in 59. Akira, S., Uematsu, S. & or IL-10 and are regulated by Drosophila adults. Cell 86, Takeuchi, O. Pathogen IL-1beta. Nature 484, 514-518 973-983 (1996). recognition and innate immunity. Cell 124, 783-801 (2012). 54. Bourgeois, C. & Kuchler, K. Fungal pathogens-a sweet and (2006). one. Toll-like receptor 4 polymor- 1 cell-fate determination. 42 Chapter one 60. Sutmuller, R.P., den Brok, that induces the formation of activating receptor for M.H., Kramer, M., Bennink, E.J. et Birbeck granules. Immunity 12, pathogenic fungus, Malassezia. al. Toll-like receptor 2 controls 71-81 (2000). expansion and function of regulatory T cells. J. Clin. Invest. 65. Zhang, J., Zhu, J., Bu, X., 116, 485-494 (2006). Cushion, M. et al. Cdc42 and 70. Cambi, A., Gijzen, K., de Vries RhoB activation are required for l, J., Torensma, R. et al. The 61. Veldhoen, M., Hocking, R.J., mannose receptor-mediated C-type lectin DC-SIGN (CD209) Atkins, C.J., Locksley, R.M. & phagocytosis by human is an antigen-uptake receptor Stockinger, B. TGFbeta in the alveolar macrophages. Mol. Biol. for Candida albicans on context of an inflammatory Cell 16, 824-834 (2005). dendritic cells. Eur. J. Immunol. cytokine milieu supports de 33, 532-538 (2003). novo differentiation of 66. Cambi, A., Netea, M.G., IL-17-producing T cells. Mora-Montes, H.M., Gow, N.A. et 71. Heinsbroek, S.E., Taylor, P.R., Immunity 24, 179-189 (2006). al. Dendritic cell interaction Martinez, F.O., Martinez- with Candida albicans critically Pomares, L. et al. Stage-specific 62. Gringhuis, S.I., den Dunnen, depends on N-linked mannan. J. sampling by pattern recogni- J., Litjens, M., van Het Hof, B. et al. Biol. Chem. 283, 20590-20599 tion receptors during Candida C-type lectin DC-SIGN (2008). albicans phagocytosis. PLoS Pathog. 4, e1000218 (2008). modulates Toll-like receptor signaling via Raf-1 kinase-de- 67. Taylor, P.R., Tsoni, S.V., pendent acetylation of Willment, J.A., Dennehy, K.M. et 72. LeibundGut-Landmann, S., transcription factor NF-kappaB. al. Dectin-1 is required for Gross, O., Robinson, M.J., Osorio, Immunity 26, 605-616 (2007). 63. de Jong, M.A., Vriend, L.E., beta-glucan recognition and F. et al. Syk- and CARD9- control of fungal infection. Nat. dependent coupling of innate Immunol. 8, 31-38 (2007). Theelen, B., Taylor, M.E. et al. immunity to the induction of T helper cells that produce C-type lectin Langerin is a 68. Sato, K., Yang, X.L., Yudate, T., interleukin 17. Nat. Immunol. 8, 630-638 (2007). beta-glucan receptor on human Chung, J.S. et al. Dectin-2 is a Langerhans cells that recog- pattern recognition receptor for nizes opportunistic and fungi that couples with the Fc 73. Robinson, M.J., Osorio, F., pathogenic fungi. Mol. receptor gamma chain to Rosas, M., Freitas, R.P. et al. Immunol. 47, 1216-1225 (2010). induce innate immune Dectin-2 is a Syk-coupled responses. J. Biol. Chem. 281, pattern recognition receptor 38854-38866 (2006). crucial for Th17 responses to K. et al. Langerin, a novel C-type 69. Yamasaki, S., Matsumoto, M., 206, 2037-2051 (2009). lectin specific to Langerhans Takeuchi, O., Matsuzawa, T. et al. cells, is an endocytic receptor C-type lectin Mincle is an 64. Valladeau, J., Ravel, O., Introduction Proc. Natl. Acad. Sci. U. S. A. 106, 1897-1902 (2009). Dezutter-Dambuyant, C., Moore, fungal infection. J. Exp. Med. 43 C-type lectin receptors orchestrate antifungal immunity 74. Gringhuis, S.I., den Dunnen, Spriel, A.B. et al. Early stop 83. Saijo, S., Ikeda, S., Yamabe, K., J., Litjens, M., van der Vlist, M. et polymorphism in human Kakuta, S. et al. Dectin-2 al. Dectin-1 directs T helper cell DECTIN-1 is associated with recognition of alpha-mannans differentiation by controlling increased candida colonization and induction of Th17 cell noncanonical NF-kappaB in hematopoietic stem cell differentiation is essential for activation through Raf-1 and transplant recipients. Clin. host defense against Candida Syk. Nat. Immunol. 10, 203-213 Infect. Dis. 49, 724-732 (2009). albicans. Immunity 32, 681-691 (2009). (2010). 79. Glocker, E.O., Hennigs, A., 75. Ferwerda, B., Ferwerda, G., Nabavi, M., Schaffer, A.A. et al. A 84. Wells, C.A., Salvage-Jones, Plantinga, T.S., Willment, J.A. et homozygous CARD9 mutation J.A., Li, X., Hitchens, K. et al. The al. Human dectin-1 deficiency in a family with susceptibility to macrophage-inducible C-type and mucocutaneous fungal fungal infections. N. Engl. J. Med. lectin, mincle, is an essential infections. N. Engl. J. Med. 361, 361, 1727-1735 (2009). component of the innate 80. Drewniak, A.A., Gazendam, albicans. J. Immunol. 180, 76. Chai, L.Y., de Boer, M.G., van R.P., Tool, A.T., van Houdt, M. et al. 7404-7413 (2008). der Velden, W.J., Plantinga, T.S. Invasive fungal infection and 1760-1767 (2009). immune response to Candida et al. The Y238X stop codon impaired neutrophil killing in 85. Sousa Mda, G., Reid, D.M., polymorphism in the human human CARD9 deficiency. Schweighoffer, E., Tybulewicz, beta-glucan receptor dectin-1 Blood (2013). V. et al. Restoration of pattern and susceptibility to invasive recognition receptor costimula81. Strasser, D., Neumann, K., tion to treat chromoblastomy- Bergmann, H., Marakalala, M.J. cosis, a chronic fungal infection et al. Syk kinase-coupled C-type of the skin. Cell host & microbe 77. Cunha, C., Di Ianni, M., Bozza, lectin receptors engage protein 9, 436-443 (2011). S., Giovannini, G. et al. Dectin-1 kinase C-sigma to elicit Card9 Y238X polymorphism adaptor-mediated innate 86. Gringhuis, S.I., den Dunnen, associates with susceptibility to immunity. Immunity 36, 32-42 J., Litjens, M., van der Vlist, M. & invasive aspergillosis in (2012). Geijtenbeek, T.B. Carbohydrate- 736-743 (2011). specific signaling through the hematopoietic transplantation through impairment of both 82. Yamasaki, S., Ishikawa, E., DC-SIGN signalosome tailors recipient- and donor-dependent Sakuma, M., Hara, H. et al. Mincle immunity to Mycobacterium mechanisms of antifungal is an ITAM-coupled activating tuberculosis, HIV-1 and immunity. Blood 116, 5394- receptor that senses damaged Helicobacter pylori. Nat. cells. Nat. Immunol. 9, 1179- Immunol. 10, 1081-1088 (2009). 5402 (2010). 1188 (2008). 78. Plantinga, T.S., van der 87. Willment, J.A., Marshall, A.S., Velden, W.J., Ferwerda, B., van Reid, D.M., Williams, D.L. et al. one. aspergillosis. J. Infect. Dis. 203, 44 The human beta-glucan Chapter one fungal pathogen Cryptococcus IL-1beta via a noncanonical receptor is widely expressed neoformans. Infect. Immun. 77, caspase-8 inflammasome. Nat. and functionally equivalent to 3491-3500 (2009). Immunol. 13, 246-254 (2012). murine Dectin-1 on primary cells. Eur. J. Immunol. 35, 93. Sorgi, C.A., Secatto, A., 98. Wheeler, R.T., Kombe, D., 1539-1547 (2005). Fontanari, C., Turato, W.M. et al. Agarwala, S.D. & Fink, G.R. Histoplasma capsulatum cell Dynamic, morphotype-specific 88. Martin, B., Hirota, K., Cua, D.J., wall {beta}-glucan induces lipid Candida albicans beta-glucan Stockinger, B. & Veldhoen, M. body formation through CD18, exposure during infection and Interleukin-17-producing TLR2, and dectin-1 receptors: drug treatment. PLoS Pathog. 4, gammadelta T cells selectively correlation with leukotriene B4 e1000227 (2008). expand in response to generation and role in HIV-1 pathogen products and infection. J. Immunol. 182, 99. Blander, J.M. & Sander, L.E. environmental signals. 4025-4035 (2009). Beyond pattern recognition: 94. Steele, C., Marrero, L., Swain, scaling the microbial threat. Nat. Immunity 31, 321-330 (2009). five immune checkpoints for 89. Steele, C., Rapaka, R.R., Metz, S., Harmsen, A.G. et al. Alveolar Rev. Immunol. 12, 215-225 A., Pop, S.M. et al. The beta-glu- macrophage-mediated killing (2012). can receptor dectin-1 recog- of Pneumocystis carinii f. sp. nizes specific morphologies of muris involves molecular Aspergillus fumigatus. PLoS recognition by the Dectin-1 K.J. Complementation of a Pathog. 1, e42 (2005). beta-glucan receptor. J. Exp. capsule-deficient mutation of Med. 198, 1677-1688 (2003). Cryptococcus neoformans 90. Brown, G.D., Herre, J., restores its virulence. Mol. Cell. Williams, D.L., Willment, J.A. et 95. Viriyakosol, S., Jimenez al. Dectin-1 mediates the Mdel, P., Gurney, M.A., biological effects of beta-glu- Ashbaugh, M.E. & Fierer, J. 101. Aimanianda, V., Bayry, J., cans. J. Exp. Med. 197, 1119- Dectin-1 is required for Bozza, S., Kniemeyer, O. et al. 1124 (2003). resistance to coccidioidomyco- Surface hydrophobin prevents sis in mice. mBio 4(2013). immune recognition of 91. Viriyakosol, S., Fierer, J., Biol. 14, 4912-4919 (1994). airborne fungal spores. Nature Brown, G.D. & Kirkland, T.N. 96. Yadav, M. & Schorey, J.S. The Innate immunity to the beta-glucan receptor dectin-1 460, 1117-1121 (2009). pathogenic fungus functions together with TLR2 102. Ariizumi, K., Shen, G.L., Coccidioides posadasii is to mediate macrophage Shikano, S., Xu, S. et al. Identi- dependent on Toll-like receptor activation by mycobacteria. fication of a novel, dendritic 2 and Dectin-1. Infect. Immun. Blood 108, 3168-3175 (2006). cell-associated molecule, 73, 1553-1560 (2005). Introduction 100. Chang, Y.C. & Kwon-Chung, dectin-1, by subtractive cDNA 97. Gringhuis, S.I., Kaptein, T.M., cloning. J. Biol. Chem. 275, 92. Giles, S.S., Dagenais, T.R., Wevers, B.A., Theelen, B. et al. 20157-20167 (2000). Botts, M.R., Keller, N.P. & Hull, Dectin-1 is an extracellular C.M. Elucidating the pathogene- pathogen sensor for the 103. Kerrigan, A.M. & Brown, G.D. sis of spores from the human induction and processing of Syk-coupled C-type lectin 45 C-type lectin receptors orchestrate antifungal immunity receptors that mediate cellular Calcineurin regulates innate infection of dendritic cells. Nat. activation via single tyrosine antifungal immunity in Immunol. 11, 419-426 (2010). based activation motifs. neutrophils. J. Exp. Med. 207, Immunol. Rev. 234, 335-352 923-931 (2010). 115. Soilleux, E.J., Morris, L.S., 110. Ma, J., Becker, C., Lowell, C.A. Constitutive and induced (2010). Leslie, G., Chehimi, J. et al. 104. Rogers, N.C., Slack, E.C., & Underhill, D.M. Dectin-1- expression of DC-SIGN on Edwards, A.D., Nolte, M.A. et al. triggered recruitment of light dendritic cell and macrophage Syk-dependent cytokine chain 3 protein to phagosomes subpopulations in situ and in induction by Dectin-1 reveals a facilitates major histocompati- vitro. J. Leukoc. Biol.71, 445-457 novel pattern recognition bility complex class II (2002). pathway for C type lectins. presentation of fungal-derived Immunity 22, 507-517 (2005). antigens. J. Biol. Chem. 287, 116. Koppel, E.A., van Gisbergen, 34149-34156 (2012). K.P., Geijtenbeek, T.B. & van 105. Hughes, C.E., Pollitt, A.Y., Kooyk, Y. Distinct functions of Mori, J., Eble, J.A. et al. CLEC-2 111. Gross, O., Gewies, A., Finger, activates Syk through K., Schafer, M. et al. Card9 L-SIGN (DC-SIGNR) and dimerization. Blood 115, controls a non-TLR signalling mSIGNR1 in pathogen 2947-2955 (2010). pathway for innate anti-fungal recognition and immune DC-SIGN and its homologues immunity. Nature 442, 651-656 regulation. Cell. Microbiol. 7, (2006). 157-165 (2005). tyrosine kinase: a crucial player 112. Rawlings, D.J., Sommer, K. & 117. van den Berg, L.M., in diverse biological functions. Moreno-Garcia, M.E. The Gringhuis, S.I. & Geijtenbeek, T.B. Nat. Rev. Immunol. 10, 387-402 CARMA1 signalosome links the An evolutionary perspective on (2010). signalling machinery of C-type lectins in Infect. Immun.. adaptive and innate immunity Ann. N. Y. Acad. Sci.1253, 107. Goodridge, H.S., Reyes, C.N., in lymphocytes. Nat. Rev. 149-158 (2012). Becker, C.A., Katsumoto, T.R. et Immunol. 6, 799-812 (2006). 106. Mocsai, A., Ruland, J. & Tybulewicz, V.L. The SYK immune receptor Dectin-1 113. Kawadler, H., Gantz, M.A., T.B., van Vliet, S.J., Wijers, M. et al. upon formation of a ‘phagocytic Riley, J.L. & Yang, X. The The dendritic cell-specific synapse’. Nature 472, 471-475 paracaspase MALT1 controls adhesion receptor DC-SIGN (2011). caspase-8 activation during internalizes antigen for lymphocyte proliferation. Mol. presentation to T cells. J. 108. Goodridge, H.S., Wolf, A.J. & Cell 31, 415-421 (2008). Underhill, D.M. Beta-glucan Immunol. 168, 2118-2126 (2002). recognition by the innate 114. Gringhuis, S.I., van der Vlist, immune system. Immunol. Rev. M., van den Berg, L.M., den 230, 38-50 (2009). Dunnen, J. et al. HIV-1 exploits Dominguez-Soto, A., Ancochea, innate signaling by TLR8 and J., Jimenez-Heffernan, J.A. et al. DC-SIGN for productive Dendritic cell-specific 109. Greenblatt, M.B., Aliprantis, A., Hu, B. & Glimcher, L.H. 119. Serrano-Gomez, D., intercellular adhesion one. 118. Engering, A., Geijtenbeek, al. Activation of the innate 46 Chapter one molecule 3-grabbing noninteg- 124. McGreal, E.P., Rosas, M., endocytic receptor. Eur. J. rin mediates binding and Brown, G.D., Zamze, S. et al. The Immunol. 34, 210-220 (2004). internalization of Aspergillus carbohydrate-recognition fumigatus conidia by dendritic domain of Dectin-2 is a C-type 129. Graham, L.M., Gupta, V., cells and macrophages. J. lectin with specificity for high Schafer, G., Reid, D.M. et al. The Immunol. 173, 5635-5643 mannose. Glycobiology 16, C-type lectin receptor CLECSF8 (2004). 422-430 (2006). (CLEC4D) is expressed by 120. Mansour, M.K., Latz, E. & 125. Barrett, N.A., Maekawa, A., cellular activation through Syk Levitz, S.M. Cryptococcus Rahman, O.M., Austen, K.F. & kinase. J. Biol. Chem. 287, neoformans glycoantigens are Kanaoka, Y. Dectin-2 recogni- 25964-25974 (2012). myeloid cells and triggers captured by multiple lectin tion of house dust mite triggers receptors and presented by cysteinyl leukotriene genera- 130. Zhu, L.L., Zhao, X.Q., Jiang, dendritic cells. J. Immunol. 176, tion by dendritic cells. J. C., You, Y. et al. C-type lectin Immunol. 182, 1119-1128 receptors Dectin-3 and Dectin-2 (2009). form a heterodimeric pat- 3053-3061 (2006). 121. Takashima, M., Hamamoto, M. & Nakase, T. Taxonomic tern-recognition receptor for 126. Ishikawa, T., Itoh, F., Yoshida, host defense against fungal significance of fucose in the S., Saijo, S. et al. Identification of infection. Immunity 39, class Urediniomycetes: Distinct Ligands for the C-type 324-334 (2013). distribution of fucose in cell Lectin Receptors Mincle and wall and phylogeny of Dectin-2 in the Pathogenic 131. Clarke, D.L., Davis, N.H., urediniomycetous yeasts. Syst. Fungus Malassezia. Cell host & Campion, C.L., Foster, M.L. et al. Appl. Microbiol. 23, 63-70 (2000). microbe 13, 477-488 (2013). Dectin-2 sensing of house dust mite is critical for the initiation 122. Ariizumi, K., Shen, G.L., 127. Ritter, M., Gross, O., Kays, S., of airway inflammation. Shikano, S., Ritter, R., 3rd et al. Ruland, J. et al. Schistosoma Mucosal Immunol. (2013). Cloning of a second dendritic mansoni triggers Dectin-2, cell-associated C-type lectin which activates the Nlrp3 132. Vijayan, D., Radford, K.J., (dectin-2) and its alternatively inflammasome and alters Beckhouse, A.G., Ashman, R.B. & spliced isoforms. J. Biol. Chem. adaptive immune responses. Wells, C.A. Mincle polarizes 275, 11957-11963 (2000). Proc. Natl. Acad. Sci. U.S.A. 107, human monocyte and 20459-20464 (2010). Introduction 123. Kanazawa, N., Tashiro, K., neutrophil responses to Candida albicans. Immunol. Inaba, K., Lutz, M.B. & Miyachi, Y. 128. Arce, I., Martinez-Munoz, L., Molecular cloning of human Roda-Navarro, P. & Fernandez- dectin-2. J. Invest. Dermatol. 122, Ruiz, E. The human C-type 133. Bugarcic, A., Hitchens, K., lectin CLECSF8 is a novel Beckhouse, A.G., Wells, C.A. et al. monocyte/macrophage Human and mouse macro- 1522-1524 (2004). Cell Biol. 90, 889-895 (2012). 47 C-type lectin receptors orchestrate antifungal immunity phage-inducible C-type lectin 138. Lang, R. Recognition of the rial cord factor. Immunity 38, (Mincle) bind Candida albicans. mycobacterial cord factor by 1050-1062 (2013). Glycobiology 18, 679-685 Mincle: relevance for granu- (2008). loma formation and resistance 143. Lobato-Pascual, A., Saether, to tuberculosis. Front. Immunol. P.C., Fossum, S., Dissen, E. & 134. Ishikawa, E., Ishikawa, T., 4, 5 (2013). Daws, M.R. Mincle, the receptor Morita, Y.S., Toyonaga, K. et al. for mycobacterial cord factor, Direct recognition of the 139. Sharma, A., Steichen, A.L., forms a functional receptor mycobacterial glycolipid, Jondle, C.N., Mishra, B.B. & complex with MCL and trehalose dimycolate, by C-type Sharma, J. Protective role of FcepsilonRI-gamma. Eur. J. lectin Mincle. J. Exp. Med. 206, Mincle in bacterial pneumonia Immunol. (2013). by regulation of neutrophil mediated phagocytosis and 144. Suzuki, Y., Nakano, Y., 135. Schoenen, H., Bodendorfer, extracellular trap formation. J. Mishiro, K., Takagi, T. et al. B., Hitchens, K., Manzanero, S. et Infect. Dis. (2013). Involvement of Mincle and Syk al. Cutting edge: Mincle is in the changes to innate essential for recognition and 140. Furukawa, A., immunity after ischemic stroke. adjuvanticity of the mycobacte- Kamishikiryo, J., Mori, D., Sci. Rep. 3, 3177 (2013). rial cord factor and its synthetic Toyonaga, K. et al. Structural analog trehalose-dibehenate. J. analysis for glycolipid 145. Sancho, D. & Reis, E.S.C. Immunol. 184, 2756-2760 recognition by the C-type Sensing of cell death by (2010). 136. Desel, C., Werninghaus, K., lectins Mincle and MCL. Proc. myeloid C-type lectin receptors. Natl. Acad. Sci. U.S.A. 110, Curr. Opin. Immunol. (2013). 17438-17443 (2013). 146. Bourgeois, C., Majer, O., Ritter, M., Jozefowski, K. et al. The Mincle-activating adjuvant 141. Feinberg, H., Jegouzo, S.A., Frohner, I.E., Lesiak-Markowicz, TDB induces MyD88- Rowntree, T.J., Guan, Y. et al. I. et al. Conventional dendritic dependent Th1 and Th17 Mechanism for recognition of cells mount a type I IFN responses through IL-1R an unusual mycobacterial response against Candida spp. signaling. PLoS One 8, e53531 glycolipid by the macrophage requiring novel phagosomal receptor mincle. J. Biol. Chem. TLR7-mediated IFN-beta 288, 28457-28465 (2013). signaling. J. Immunol. 186, (2013). 137. Heitmann, L., Schoenen, H., 3104-3112 (2011). Ehlers, S., Lang, R. & Holscher, C. 142. Miyake, Y., Toyonaga, K., Mincle is not essential for Mori, D., Kakuta, S. et al. C-type 147. Biondo, C., Signorino, G., controlling Mycobacterium lectin MCL is an FcRgamma- Costa, A., Midiri, A. et al. tuberculosis infection. coupled receptor that mediates Recognition of yeast nucleic Immunobiology 218, 506-516 the adjuvanticity of mycobacte- acids triggers a host-protective (2013). type I interferon response. Eur. one. 2879-2888 (2009). 48 Chapter one J. Immunol. 41, 1969-1979 globulin production. J. Exp. Med. 161. Palm, N.W. & Medzhitov, R. (2011). 192, 1545-1552 (2000). Pattern recognition receptors and control of adaptive 148. del Fresno, C., Soulat, D., 153. O’Shea, J.J. & Paul, W.E. immunity. Immunol. Rev. 227, Roth, S., Blazek, K. et al. Mechanisms underlying 221-233 (2009). Interferon-beta production via lineage commitment and 162. Kawai, T. & Akira, S. Toll-like Dectin-1-Syk-IRF5 signaling in plasticity of helper CD4+ T cells. dendritic cells is crucial for Science 327, 1098-1102 (2010). receptors and their crosstalk immunity to C. albicans. 154. Reiner, S.L. Development in with other innate receptors in Immunity 38, 1176-1186 (2013). motion: helper T cells at work. Infect. Immun.. Immunity 34, Cell 129, 33-36 (2007). 637-650 (2011). Kumar, V., Johnson, M.D. et al. 155. Abbas, A.K., Murphy, K.M. & 163. Drickamer, K. C-type Functional genomics identifies Sher, A. Functional diversity of lectin-like domains. Curr. Opin. type I interferon pathway as helper T lymphocytes. Nature Struct. Biol. 9, 585-590 (1999). central for host defense against 383, 787-793 (1996). 149. Smeekens, S.P., Ng, A., 164. Zhang, J.G., Czabotar, P.E., Candida albicans. Nat. Commun. 4, 1342 (2013). 156. Sakaguchi, S., Miyara, M., Policheni, A.N., Caminschi, I. et Costantino, C.M. & Hafler, D.A. al. The dendritic cell receptor 150. Mosmann, T.R., Cherwinski, FOXP3+ regulatory T cells in Clec9A binds damaged cells via H., Bond, M.W., Giedlin, M.A. & the human immune system. exposed actin filaments. Coffman, R.L. Two types of Nat. Rev. Immunol. 10, 490-500 Immunity 36, 646-657 (2012). murine helper T cell clone. I. (2010). 165. Ahrens, S., Zelenay, S., Definition according to profiles of lymphokine activities and 157. Crotty, S. Follicular helper Sancho, D., Hanc, P. et al. F-actin secreted proteins. J. Immunol. CD4 T cells (TFH). Annu. Rev. is an evolutionarily conserved 136, 2348-2357 (1986). Immunol. 29, 621-663 (2011). damage-associated molecular 151. Harrington, L.E., Hatton, 158. Zhang, N. & Bevan, M.J. a receptor for dead cells. Immunity 36, 635-645 (2012). pattern recognized by DNGR-1, R.D., Mangan, P.R., Turner, H. et CD8(+) T cells: foot soldiers of al. Interleukin 17-producing the immune system. Immunity CD4+ effector T cells develop 35, 161-168 (2011). via a lineage distinct from the T The C-type lectin-like domain helper type 1 and 2 lineages. 159. Pepper, M. & Jenkins, M.K. superfamily. FEBS J. 272, Nat. Immunol. 6, 1123-1132 Origins of CD4(+) effector and 6179-6217 (2005). (2005). central memory T cells. Nat. Immunol. 12, 467-471 (2011). 152. Breitfeld, D., Ohl, L., Kremmer, E., Ellwart, J. et al. Introduction 166. Zelensky, A.N. & Gready, J.E. 167. Wen, H., Miao, E.A. & Ting, J.P. Mechanisms of NOD-like 160. Bevan, M.J. Understand receptor-associated inflam- Follicular B helper T cells memory, design better masome activation. Immunity express CXC chemokine vaccines. Nat. Immunol. 12, 39, 432-441 (2013). receptor 5, localize to B cell 463-465 (2011). follicles, and support immuno- C-type lectin receptors orchestrate antifungal immunity 49 168. Loo, Y.M. & Gale, M., Jr. Immune signaling by RIG-I-like receptors. Immunity 34, 680-692 (2011). 169. Broz, P. & Monack, D.M. Newly described pattern recognition receptors team up against intracellular pathogens. Nat. Rev. Immunol. 13, 551-565 (2013). 170. Sun, L., Deng, L., Ea, C.K., Xia, Z.P. & Chen, Z.J. The TRAF6 ubiquitin ligase and TAK1 kinase mediate IKK activation by BCL10 and MALT1 in T lymphocytes. Mol. Cell 14, one. 289-301 (2004).